A Cytochrome P450 Immunochemically Related to P450_c,d(P450I) Localized to the Smooth Microsomes and Inner Zone of the Guinea Pig Adrenal*

Abstract

In addition to their capacity for steroid synthesis, guinea pig adrenal microsomes have a well documented ability to metabolize foreign compounds. The capacity for metabolism of foreign compounds is localized to the smooth endoplasmic reticulum-filled cells of the inner zone. However, it has not been clear whether they possess cytochrome P450(s) specific for this function, distinct from the two known steroid hydroxylases, P450(21) and P450(17)alpha. Multiple prominent protein bands in the mol wt range of known cytochrome P450s are seen on sodium dodecyl sulfate gels of guinea pig adrenal microsomes. Most are more intense in smooth microsomes, where the concentration of cytochrome P450 is highest. However, one band (52K) appears unique to the smooth microsomes. This band is also characteristic of microsomes obtained from the inner zone. This protein and two others (54K and 50K) are concentrated in the membrane pellet after carbonate treatment of the microsomes, indicating that they are integral membrane proteins. All three decrease in intensity after treatment of the animals with spironolactone, a compound known to cause depletion of adrenal cytochrome P450s. On Western blots of microsomal proteins the 54K and 50K proteins react with antibodies specific for P450(17) alpha and P450(21), respectively. The 52K protein, characteristic of the smooth microsomes and inner zone, does not react with anti-P450(21) or anti-P450(17) alpha, but does react with polyclonal antibody raised against microsomal cytochrome P450s induced by methylcholanthrene in rat liver (P450c,d). These results suggest that there is at least one additional cytochrome P450 in adrenal microsomes which is immunochemically distinct from P450(21) and P450(17) alpha. Its localization to the smooth microsomes and inner zone microsomes correlates with the high activity for ethylmorphine metabolism detected in these fractions. This suggests that this cytochrome P450, which is immunochemically related to members of the P450I subfamily, may be associated with the ability of guinea pig adrenal microsomes to metabolize foreign compounds.